A Finnigan LTQ linear ion trap mass spectrometer equipped with a nanospray interface was used as the detector for the peptides that eluted from the RPC column. Full scan mass spectra were collected in profile mode and MS/MS spectra in centroid mode.
Detection of significantly varying peptides using DeCyder MS
Detection, profile comparison, background subtraction, and quantitation were done on the full scan precursor mass spectra in fully automatic mode using DeCyder MS (1).
The PepDetect module of DeCyder MS was used for automated peptide detection, charge-state assignments, deconvolution, and quantitation based on MS signal intensities of individual LC-MS analyses.
The PepMatch module was used to match peptides in different intensity maps from the different runs, which resulted in a quantitative comparison including statistical evaluation. The background intensity was used for normalization (no internal standards were added to the samples). Peptides were identified using intact masses by exporting the MS/MS files into TurboSEQUEST protein identification software and subsequently importing TurboSEQUEST search results into the PepMatch module. The peptide matches were filtered based on cross-correlation scores (Xcorr) of 1.5, 2, and 2.5 for charge states 1+, 2+, and 3+, respectively.
Results and discussion
Protein prefractionation of allergen samples
The two allergen samples, Ole_e1_"lite" and Ole_e1_"me